Biosynthetic Pathway of Cephabacins in Lysobacter lactamgenus: Molecular and Biochemical Characterization of the Upstream Region of the Gene Clusters for Engineering of Novel Antibiotics

AbstractCytochrome P450 monooxygenases (P450s) play crucial roles in the cell metabolism and provide an unsurpassed diversity of catalysed reactions. Here, we report the identification and biochemical characterization of two P450s from Arthrobacter sp., a Gram-positive organism known to degrade the opium alkaloid papaverine. Combining phylogenetic and genomic analysis suggested physiological roles for P450s in metabolism and revealed potential gene clusters with redox partners facilitating the reconstitution of the P450 activities in vitro. CYP1232F1 catalyses the para demethylation of 3,4-dimethoxyphenylacetic acid to homovanillic acid while CYP1232A24 continues demethylation to 3,4-dihydroxyphenylacetic acid. Interestingly, the latter enzyme is also able to perform both demethylation steps with preference for the meta position. The crystal structure of CYP1232A24, which shares only 29% identity to previous published structures of P450s helped to rationalize the preferred demethylation specificity for the meta position and also the broader substrate specificity profile. In addition to the detailed characterization of the two P450s using their physiological redox partners, we report the construction of a highly active whole-cell Escherichia coli biocatalyst expressing CYP1232A24, which formed up to 1.77 g l−1 3,4-dihydroxyphenylacetic acid. Our results revealed the P450s’ role in the metabolic pathway of papaverine enabling further investigation and application of these biocatalysts.

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Inhibitors of Pantothenate Kinase: Novel Antibiotics for Staphylococcal Infections

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.47.6.2051-2055.2003 ◽

2003 ◽

Vol 47 (6) ◽

pp. 2051-2055 ◽

Cited By ~ 61

Author(s):

Anthony E. Choudhry ◽

Tracy L. Mandichak ◽

John P. Broskey ◽

Richard W. Egolf ◽

Cynthia Kinsland ◽

...

Keyword(s):

Molecular Weight ◽

Staphylococcus Aureus ◽

Biosynthetic Pathway ◽

Coenzyme A ◽

Low Molecular Weight ◽

Pantothenate Kinase ◽

Staphylococcal Infections ◽

Low Molecular Weight Compounds ◽

Novel Antibiotics

ABSTRACT Pantothenate kinase (CoaA) catalyzes the first step of the coenzyme A biosynthetic pathway. Here we report the identification of the Staphylococcus aureus coaA gene and characterization of the enzyme. We have also identified a series of low-molecular-weight compounds which are effective inhibitors of S. aureus CoaA.

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Biochemical Characterization of the CDP-D-Arabinitol Biosynthetic Pathway in Streptococcus pneumoniae 17F

Journal of Bacteriology ◽

10.1128/jb.06487-11 ◽

2012 ◽

Vol 194 (8) ◽

pp. 1868-1874 ◽

Cited By ~ 3

Author(s):

Q. Wang ◽

Y. Xu ◽

A. V. Perepelov ◽

Y. A. Knirel ◽

P. R. Reeves ◽

...

Keyword(s):

Streptococcus Pneumoniae ◽

Biosynthetic Pathway ◽

Biochemical Characterization

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Genome mining of novel rubiginones from Streptomyces sp. CB02414 and characterization of the post-PKS modification steps in rubiginone biosynthesis

Microbial Cell Factories ◽

10.1186/s12934-021-01681-5 ◽

2021 ◽

Vol 20 (1) ◽

Author(s):

Jingyan Zhang ◽

Ying Sun ◽

Yeji Wang ◽

Xin Chen ◽

Lu Xue ◽

...

Keyword(s):

Gene Cluster ◽

Biosynthetic Pathway ◽

Scale Up ◽

Genome Mining ◽

Gene Clusters ◽

Combinatorial Biosynthesis ◽

Wild Type ◽

Aromatic Polyketides ◽

A Genome

Abstract Background Rubiginones belong to the angucycline family of aromatic polyketides, and they have been shown to potentiate the vincristine (VCR)-induced cytotoxicity against VCR-resistant cancer cell lines. However, the biosynthetic gene clusters (BGCs) and biosynthetic pathways for rubiginones have not been reported yet. Results In this study, based on bioinformatics analysis of the genome of Streptomyces sp. CB02414, we predicted the functions of the two type II polyketide synthases (PKSs) BGCs. The rub gene cluster was predicted to encode metabolites of the angucycline family. Scale-up fermentation of the CB02414 wild-type strain led to the discovery of eight rubiginones, including five new ones (rubiginones J, K, L, M, and N). Rubiginone J was proposed to be the final product of the rub gene cluster, which features extensive oxidation on the A-ring of the angucycline skeleton. Based on the production profiles of the CB02414 wild-type and the mutant strains, we proposed a biosynthetic pathway for the rubiginones in CB02414. Conclusions A genome mining strategy enabled the efficient discovery of new rubiginones from Streptomyces sp. CB02414. Based on the isolated biosynthetic intermediates, a plausible biosynthetic pathway for the rubiginones was proposed. Our research lays the foundation for further studies on the mechanism of the cytochrome P450-catalyzed oxidation of angucyclines and for the generation of novel angucyclines using combinatorial biosynthesis strategies.

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Activation and Identification of a Griseusin Cluster in Streptomyces sp. CA-256286 by Employing Transcriptional Regulators and Multi-Omics Methods

Molecules ◽

10.3390/molecules26216580 ◽

2021 ◽

Vol 26 (21) ◽

pp. 6580

Author(s):

Charlotte Beck ◽

Tetiana Gren ◽

Francisco Javier Ortiz-López ◽

Tue Sparholt Jørgensen ◽

Daniel Carretero-Molina ◽

...

Keyword(s):

Biosynthetic Pathway ◽

Genome Mining ◽

Gene Clusters ◽

Transcriptional Regulators ◽

Biosynthetic Gene ◽

Biosynthetic Gene Clusters ◽

Heterologous Host ◽

Streptomyces Sp ◽

Identification And Characterization

Streptomyces are well-known producers of a range of different secondary metabolites, including antibiotics and other bioactive compounds. Recently, it has been demonstrated that “silent” biosynthetic gene clusters (BGCs) can be activated by heterologously expressing transcriptional regulators from other BGCs. Here, we have activated a silent BGC in Streptomyces sp. CA-256286 by overexpression of a set of SARP family transcriptional regulators. The structure of the produced compound was elucidated by NMR and found to be an N-acetyl cysteine adduct of the pyranonaphtoquinone polyketide 3′-O-α-d-forosaminyl-(+)-griseusin A. Employing a combination of multi-omics and metabolic engineering techniques, we identified the responsible BGC. These methods include genome mining, proteomics and transcriptomics analyses, in combination with CRISPR induced gene inactivations and expression of the BGC in a heterologous host strain. This work demonstrates an easy-to-implement workflow of how silent BGCs can be activated, followed by the identification and characterization of the produced compound, the responsible BGC, and hints of its biosynthetic pathway.

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Discovery and Biocatalytic Application of a PLP-Dependent Amino Acid γ-Substitution Enzyme that Catalyzes C-C Bond Formation

10.26434/chemrxiv.12052410 ◽

2020 ◽

Author(s):

Mengbin Chen ◽

Chun-Ting Liu ◽

Yi Tang

Keyword(s):

Amino Acids ◽

Biosynthetic Pathway ◽

Pyridoxal Phosphate ◽

Biochemical Characterization ◽

Indole Alkaloid ◽

Building Blocks ◽

Nucleophilic Attack ◽

Keto Esters ◽

Key Enzyme

Pyridoxal phosphate (PLP)-dependent enzymes can catalyze various transformations of amino acids at alpha, beta, and gamma positions. These versatile enzymes are prominently involved in the biosynthesis of nonproteinogenic amino acids as building blocks of natural products, and are attractive biocatalysts. Here, we report the discovery of a two-step enzymatic synthesis of (2S, 6S)-6-methyl pipecolate 1, from the biosynthetic pathway of indole alkaloid citrinadin. The key enzyme CndF is PLP-dependent and catalyzes synthesis of (S)-2-amino-6-oxoheptanoate 3 that is in equilibrium with the cyclic Schiff base. The second enzyme CndE is a stereoselective imine reductase that gives 1. Biochemical characterization of CndF showed this enzyme performs gamma-elimination of O-acetyl L-homoserine to generate the vinylglycine ketimine, which is subjected to nucleophilic attack by acetoacetate to form the new Cgamma-Cdelta bond in 3 and complete the gamma-substitution reaction. CndF displays substrate promiscuity towards different beta-keto carboxylate and esters. Using a recombinant Aspergillus strain expressing CndF and CndE, feeding various alkyl-beta-keto esters led to the biosynthesis of 6-substituted L-pipecolates. The discovery of CndF expands the repertoire of reactions that can be catalyzed by PLP-dependent enzymes.

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Characterization of an Escherichia coli K12 mutant that is sensitive to chlorate when grown aerobically

Biochemical Journal ◽

10.1042/bj1760553 ◽

1978 ◽

Vol 176 (2) ◽

pp. 553-561 ◽

Cited By ~ 17

Author(s):

G Giordano ◽

L Grillet ◽

R Rosset ◽

J H Dou ◽

E Azoulay ◽

...

Keyword(s):

Escherichia Coli ◽

Biosynthetic Pathway ◽

Formate Dehydrogenase ◽

Biochemical Characterization ◽

Reductase Activity ◽

Growth Conditions ◽

Aerobic Growth ◽

Benzyl Viologen ◽

Genetic Lesion

Escherichia coli can normally grow aerobically in the presence of chlorate; however, mutants can be isolated that can no longer grow under these conditions. We present here the biochemical characterization of one such mutant and show that the primary genetic lesion occurs in the ubiquinone-8-biosynthetic pathway. As a consequence of this, under aerobic growth conditions the mutant is apparently unable to synthesize formate dehydrogenase, but can synthesize a Benzyl Viologen-dependent nitrate reductase activity. The nature of this activity is discussed.

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